Current studies have on the identification of novel antigens expressed in melanoma and renal cancers as well as the characterization of T cells that recognize epitopes of previously described antigens. In a recent studies, several novel point-mutated genes have been identified as the targets of melanoma-reactive TIL that mediated long terms clinical tumor regression following adoptive transfer. Current investigations are being carried out to develop methods to facilitate the identification of mutated gene products that can be evaluated as targets of tumor-reactive T cells. Recent studies have focused on the use of high throughput sequencing to identify all of the somatic mutations in patient tumors. A set of candidate mutated T cell epitopes were then identified using an MHC/peptide binding algorithm and tested for their ability to be recognized by patient TIL. Screening of candidate antigens identified resulted in the identification of either two or three mutated epitopes as targets of each of the three melanoma-reactive TIL that were initially evaluated using this approach. In ongoing studies, T cells that recognize additional cancers are also being evaluated for their ability to recognize candidate epitopes identified from patient tumors. These results may provide important tools for the development of effective adoptive immunotherapies for the treatment of a variety of malignancies, as well as providing potent vaccine candidates. Studies are also being carried out in collaboration with the NIH RNAi consortium to develop a novel approach to the identification of human tumor antigens. Screening assays are now being performed by transfection of tumor cells with an siRNA library targeting over 20,000 human cDNA transcripts in an attempt to down-regulate tumor recognition by autologous patient T cells. This approach, if successful, will represent a powerful method for the identification of tumor antigens, as a single set of reagents could potentially be used for screening T cells of any specificity. Additional studies have involved the identification of T cell receptors (TCRs) directed against widely shared antigens. Recent studies have focused on the identification of TCRs that recognize cancer/testis (C/T) antigens, molecules that are expressed in melanoma as well as a variety of highly prevalent malignancies such as breast and prostate cancer. A TCR that recognizes the NY-ESO-1 C/T antigen was used to transduce autologous PBMC, and results of a Phase II clinical trial published in January of 2011 indicate that approximately 50% of either melanoma or synovial cell sarcoma patients who received transudced PBMC demonstrated objective clinical responses to adoptively transferred autologous PBMC that were transduced with the NY-ESO-1 TCR. The DNA core facility provides services to many of the members of the Surgery Branch, which includes expertise and reagents used for RT-PCR, quantatitive RT-PCR, gene cloning and RNA/DNA analysis. For some of these studies, sequences were analyzed on an Applied Biosystems 3100-Avant Genetic Analyzer that is a part of the Surgery Branch DNA Sequencing Core. The FACS Core Facility is currently being utilized for the analysis of T cell populations that are administered to patients as a part of the analysis of ongoing clinical cancer adoptive immunotherapy trials. In addition, the FACS Core is utilized on a daily basis for the analysis of the results of experiments to analyze the results of in vitro experiments to examine factors that influence the phenotype and function of tumor reactive T cells as well as to carry out the separation of cells based upon their expression of a wide variety of cell surface markers. For these studies phenotypic analyses were carried out on a BD FACSCalibur, FACSCantoI and FACSCantoII, and cell separations were carried out on a BD FACSAriaII.